应用DNA条形码技术快速鉴定入境船舶上截获的昆虫蛹

目的]利用DNA条形码技术快速准确鉴定从入境船舶上截获的未知双翅目昆虫蛹。方法]将截获的5只蛹，1只蛹直接冷冻，4只孵化出成蝇；对蛹、蛹壳及孵化出的成蝇进行拍照留档。分别从蛹、蛹壳和孵化成蝇中提取基因组DNA；采用动物DNA条形码通用引物（LCO1490和HCO2198）扩增目的片段，并进行纯化、克隆测序和序列分析，与NCBI及BOLD中的序列进行比对，构建NJ树；对孵化成蝇进行形态鉴定。结果]所检测的蛹、蛹壳以及孵化成蝇的DNA条形码序列与NCBI和BOLD中大头金蝇（Chrysomyia megacephala）的序列相似度达100%；孵化成蝇与大头金蝇的形态特征相符。结论]根据测定结果，判定截获的未知双翅目昆虫蛹为大头金蝇的蛹；证明DNA条形码技术是一种鉴定未知昆虫蛹的有效手段。对于口岸截获的外来未知昆虫蛹、成虫残肢甚至蛹壳均可直接采用DNA条形码技术进行种类鉴定，大大缩短鉴定周期。

DNA Barcoding Identification of Unknown Pupa Intercepted From Entry Ship

Objective] In order to identify the unknown Diptera pupae intercepted from an entry ship rapidly and accurately with DNA barcoding. Method] Five intercepted Diptera pupae were separated into two groups, a pupa was frozen to death, four pupas were hatched to adults. Pupa, puparium and adults were photographed and saved for further references. Genomic DNA were extracted from the pupa, puparium and adults separately. Target fragments were amplified with animal DNA barcoding universal primers （LCO1490 and HCO2198）, then purified, cloned, sequenced and blasted in NCBI and BOLD, and a NJ tree was built based the original data. Results] Sequence of the DNA barcode fragment from the unknown pupa, puparium and adults were 100% identical to the published sequence of Chrysomyia megacephala in the GenBank and BOLD. Incubated adult was identified as C. megacephala morphologically. Conclusion] Based on the results, it could be concluded that the unknown diptera pupae was C. megacephala, and in addition it is affirmed again that DNA barcoding is an effective method to identify the unknown pupae. Intercepted unknown pupa, even the morphology incomplete adult caught at POE could be identified by DNA barcode directly.